Process for the preparation of amylase

ABSTRACT

Acid-active and acid-stable amylase having superior amylolytic activity under acidic conditions is obtained by culturing an amylase-producing micro-organism of the Paecilomyces subglobosum group in a nutrient medium therefor.

ite States Patent Sawada et al.

[4 1 Mar. 28, 1972 Mar. 4, 1965 PROCESS FOR THE PREPARATION 01F AMYLASE Jiro Sawada; Tetsuo Misaki; Masao Okabe; Hajime Yasui; Kazunori Hanada; Tadayasu Okazaki, all of Tokyo, Japan inventors:

Assignee: Taisho Pharmaceutical Ltd., Tokyo, Japan I Filed: Mar. 4,1966

Appl.No.: 531,816

Foreign Application Priority Data Japan ..40/12ll4 US. Cl. .l ..l95/66 R, 195/31 R, 195/62 v lint. Cl. ..Cl2d 13/10 Primary Examiner-Lionel M. Shapiro Attorney--Wenderoth, Lind & Ponack [57] ABSTRACT Acid-active and acid-stable amylase having superior amylolytic activity under acidie conditions is obtained by culturing an amylase-producing micro-organism of the Paecilomyces subglobosum group in a nutrient medium therefor.

8 Claims, 12 Drawing Figures PATENTEDMAR281972 3,652,400

' sum 1 UF 4 unit/g.

FIG. 1

unit/g.

INVENTORS JIRO SAWADA TETSUO MISAKI MASAO OKABE HAJIME YASUI KAZUNORI HANADA BY TADAYASU OKAZAKI WM, W ATTORNEYS PATENTEI] MR 2 8 I972 FIG 3 RESIDUAL ACTIVITY (/o) RESIDUAL ACTIVITY SHEET 2 [IF 4 l 1 l l l l l L 1 7 a 9 l0 (pH) INVENTORS JIRO SAWADA TETSUO MISAKI MASAO OKABE HAJIME YASUI KAZUNORI HANADA TADAYASU OKAZAKI BY WM 4 ATTORNEYS PATENTEDMAR28 I972 13, 652,400

SHEET 4 BF 4 unii/q.

unit/g.

40,000 20,000 P 1 l l 2 3 4 5 6 7 a 9 (pH) unit/g. unit/q. I60,000

' INVENTORS 9 (PH) JIRO SAWADA TETSUO MISAKI MASAO OKABE HAJIME YASUI KAZUNORl HANADA BY TADAYASU OKAZAKI PROCESS FOR THE PREPARATION OF AMYLASE This invention relates to a novel and useful amylase and to a process for the preparation of the same.

More particularly, this invention is concerned with an acidactive and stable amylase derived from a new strain of microorganism belonging to Paecilomyces subglobosum as novel and useful product and moreover with a process for the preparation of the said novel enzyme.

Various kinds of microorganism have heretofore been employed to produce various types of amylolytic enzyme and among them some strains had been extensively used in the industry, for example, strains of the yellow-greenish Aspergilli group such as Aspergillus oryzae and of the blackish Aspergilli group such as Aspergillus usamii, Aspergillus awamori and Aspergillus niger, a certain strain of the Rhizopus group, and bacterium such as Bacillus substilis.

Generally, some differences in enzymatic properties have been found between the above microbial amylase preparations.

Accordingly, it may be considered that they were used according to each characteristic property, for example, active pH range, pH-stability such as acid-stability and alkali-stability, heat-stability and amylolytic mode.

With respect to amylolytic activity, many amylase preparations derived from various microorganisms have been found to possess two kinds of typical activities, namely, dextrinogenic and saccharifying functions against starch.

Some amylase preparations possessing acid-activity and acid-stability of dextrinogenic and saccharifying functions are suitable for various uses under acidic conditions, being especially suitable as digestive enzyme which functions against dietary starch in the stomach where the gastric juice provides a strongly acidic condition.

Akaboli et al. (Symposium of Enzymatic Chemistry in Japan, Vol. 7, P. 107, 1952) reported an amylase preparation derived from Aspergillus oryzae to have dextrinogenic activity at pH ranging from 4.0 to 7.0 and to be easily inactivated at pH below 3.0, and, in Hakko Kogaku Zasshi (J. Fermentation Technology in Japan), Sakamoto et a]. demonstrated that Rhizopus group microorganisms produce an amylase with optimum dextrinogenic activity at about pH 5.0.

According to Minoda (J Agr. Chem. Soc. Japan, Vol. 29, P. 115, 1955, and Vol. 35, P. 479 and 482, 1961, Japanese Patent Published Specification No. 35-5046 and Agr. Biol. Chem., Vol. 37, No. 11, P. 809, 1963), amylases are derived from strains belonging to Aspergillus niger and among thus-obtained amylase preparations, the dextrinogenic activity is mainly indicated at pH above 4.0, the preparations being sufficiently stable under the acidic conditions.

However, commercial forms of the amylase preparations according to methods of the above, and other known commercial preparations hereinafter described, show neither desirable amylolytic activity nor stability under acidic conditions lower than pH 4.0 (cf. FIGS. 1, 2, 3 and 4 of the accompanying drawings).

In sharp contrast, the new strains, Paecilomyces subglobosum TPR-38ll employed in this invention can produce an acid-active and acid-stable amylase preparation which is stronger and more stable in dextrinogenic and saccharifying activities at an acidic pH of more than 3.0 and furthermore exhibits the desirable activities thereof at pH 2.5 (cf. tables hereinafter described).

Accordingly, the present invention provides new amylase preparations which are active at pH ranging from 2.5 to 6.0, in particular are stronger than the prior preparations at pH ranging from 2.5 to 3.5 and also are stable at pH ranging from 3.5 to 9.0, in particular more stable than the prior preparations at PH ranging from 3.5 to 5.0 in dextrinogenic and saccharifying activities (cf. FIGS. 1, 2, 3 and 4).

A new acid-stable digestive enzyme had heretofore been prepared by using a strain (TPR-220; ATCC No. 14572) belonging to the Paecilomyces varioti group, but it could not satisfactorily serve our purpose to obtain an acid-active and stable amylase preparation (cf. FIGS. 9 and 10).

As a result of the present invention, we have now discovered among the several microorganisms belonging to the Paecilomyces subglobosum group two new microorganisms producing amylase preparations which function at an acidic pH ranging from 2.5 to 4.5, the optimum pH being 4.0 to 4.5.

The new microorganisms, which are different from Paecilomyces van'oti have been designated Paecilomyces subglobosum TPR-38l0 and TPR-38l 1.

It is one object of this invention to isolate new microorganisms producing an acid-active and stable amylase preparation.

The main object of this invention is to obtain excellent enzyme having the above acid active and stable amylolytic activity by employing the new microorganisms, Paecilamyces subglobosum TPR-38 l0, TPR-381 l and other strains possessing the same biological characteristics as TPR-38l0 and TPR-38l 1.

It is still another object of this invention to obtain easily the aforesaid novel and useful amylase preparations.

The new microorganisms of this invention are isolated from wild fungi by the following screening which serves satisfactorily our purpose.

METHOD FOR THE ISOLATION OF THE STRAINS 1st step:

Petri-dishes, in which a modified Czapek-Dox agar culture medium:

carbon source: nitrogen source: inorganic salts:

0.5% ofsoluble starch;

0.5% of peptone;

0.1% of potassium dihydrogen phosphate;

0.05% of potassium chloride; 0.05% of magnesium sulphate; 0.001% of ferrous sulphate carbon source: nitrogen source: inorganic salts:

3% of glucose;

2% of peptone;

0.1% of potassium dihydrogen phosphate;

0.05% of potassium chloride; 0.05% of magnesium sulphate; 0.001% of ferrous sulphate,

at 30 C for 3 days with shaking. 3rd step:

Among the 50 selected strains which produce such acid-active and stable amylase functioning at pH 3, two microorganisms producing the strongest acid-active and more stable amylase are further selected for use in this invention.

From morphological observations, the said two strains belong to the same species and besides they have quite identical characteristics in:

i. the activities and properties of amylase which they produce in a liquid culture medium;

ii. the productivity of same pale brown pigments in the culture medium;

iii. the availabity of nitrites.

The morphological characteristics of the new discovered microorgansims, Paecilomyces subglobosum TPR-38l0 and TPR-38l l are as follows:

MORPHOLOGIES OF NEW MICROORGANISMS Colonial observations of the microorganisms were performed with the colonies produced on agar-slant plates.

Detailed morphological studies were carried out of the mycelia growth on slide-glass.

l. Colonies:

surface flat color greenish brown or yellowish brown 2. Spore forming part:

similar to genus Paecilomyces, sterigmata variously partly arising singly, partly two to four of them making a verticil, with some exceptional dense growing, giving an irregular penicillus-like appearance.

3. Sterigmata:

ample like shaped, usually with a constriction at center,

measuring 21 by 3.5 p. on an average, slightly rough surfaced.

4. Conidia:

form a long chain from the apex of sterigmata, globose or sub-globose, sometimes elliptical, measuring 5.5 by 3.6 [L on an average, a little rough surfaced.

5. Penicillus:

typical ones are not seen.

6. Metulae or branches:

not so definite or differentiated, if any, measuring 12 by 3.5 n on an average.

7. Conidiophore:

about the size of 20 by 4 p. on an average.

8. Macrospore:

sometimes seen in media, subglobular; 8 by 10 p. mostly.

9. Sclerotia,'perithecia or ascospores none.

10. Funicularly rising aerial mycelia and gelatinlike exudation as observed in the culture of Paecilomyces varioti none.

Relative to the apparently closely related species of Paecilomyces varioti, detailed studies clarify the differences between them.

MORPHOLOGICAL DIFFERENCES OF NEW STRAINS FROM PAECILOMYCES VARIOTI l. Conidia:

shape more globular, not so elliptical. surface a bit rougher.

2. Sterigmata:

shape a little constricted at center, not so mildly tapering.

PROCEDURE OF PRODUCING ACID-STABLE AMYLASE In accordance with one aspect of this invention, novel and useful amylase can be produce by culturing the new microorganisms, Paecilomyces subglobosum TPR-38l0 and TPR-38l l, in an appropriate nutrient medium in per se conventional manner.

According to this invention, there is established a modified culturing method which has led to good results from the fact that the obtainable quantity and activity of the amylase are essentially influenced by components of the culture medium employed.

Essentially, the medium contains a carbon source, a

nitrogen source and trace inorganic elements, each in appropriate quantity, as the nutrient substances.

i. Liquid culture medium Examples of more suitable carbon sources in the modified culture medium are percent of various kinds of starch or 5 percent of maltose, but glucose, sucrose, dextrine, glycerol and wheat flour in amount of 5 percent each can also be used instead of starch or maltose.

Suitable nitrogen sources are 3 percent of peptone, and subsidiarily there can be employed raw soybean flour, defatted v soybean, dried yeast, corn steep liquor in amount of 3 percent.

Suitable inorganic elements are:

Magnesium sulfate 0.051%

Ferrous sulfate 0.01%

Potassium chloride 0.1%

Potassium dihydrogen phosphate 0.2%

Liquid culture medium is made up by mixing the above components with water. (These components are employed as main liquid culture medium or liquid culture medium for seed koji).

It is desirable to adjust the pH value of the medium to pH 6.0 after each of these substances are added into the medium.

The maximum yield and high amylolytic activity of the obtainable amylase are generally obtained with culture treatments as follows:

In culturing tank,

Aeration rate 4/3 v/v/min.

Stirring 350 r.p.m.

Temperature 30 C Culturing time about to 94 hours.

The medium so prepared and treated is sterilized and inoculated with Paecilamyces subglobasum TPR-38I0 or TPR- 3811 seed culture medium pre-incubated upon the medium containing the same components as above for 70 hours, after which the cultivating (submerged culture) step is carried out for 90 to 94 hours with aeration.

ii. Solid culture medium Solid culture medium-base is made up by the following components, wheat bran:chaff:water in the ratio of 5:314. The solid culture medium so prepared is sterilized and inoculated with Paecilomyces subglobosum TPR38 10 or TPR38l 1 seed culture medium, the medium so inoculated is laid in state of pile and then cultivated at 30 C for about 70 hours. (Similarly, these components are employed as main solid culture medium or solid culture medium for seed koji.)

SEPARATION OF THE DESIRED AMYLASE Separation of a highly purified and active amylase from the cultured medium and extracted solution is carried out in accordance with the following procedure (a) or (b), adjusting the pH value to about 6.0 a. Precipitating process Precipitation of the desired amylase is facilitated by addition of hydrophilic organic solvents such as ethanol or acetone, or ammonium sulphate.

When it is desired to employ the said precipitating process, the following treatment leads to good results: addition of ethanol to 65 percent final concentration or addition of acetone to 55 percent final concentration and in case of ammonium sulphate, addition thereof to 0.8 of its saturation at the final concentration.

b. Adsorbing Process When it is desired to employ this method, adsorbents such as DEAE-Sephandex-ion exchanger, DEAE-Cellulose-ion exchanger and the like may advantageously be employed.

After adjusting the ion-exchanger with pH 7.0, 0.005 M phosphate bufier solution, a cultured medium or extracted solution of the amylase is adsorbed on the ion-exchanger.

The thus-adsorbed material is eluted with pH 4.0, 0.25 M sodium citrate buffer solution.

Thus adjusted eluate is treated by the same procedure as in the above precipitating process a) to obtain the desired enzyme.

DETERMINATION OF AMYLOLYTIC ACTIVITY The amylolytic activity of amylase prepared by this invention (Example 1, infra) is compared with that of the following commercial amylase preparations A, B, C and D.

Amylase preparation 8 Amylase preparation C Amylase preparation D The results of the determinations are shown graphically in the accompanying FIGS. 1 to 12 and are further explained in the following preliminary explanations and in paragraphs 1 to PRELIMINARY EXPLANATIONS FIG. 1 illustrates the activity of dextrinogenic power of the aforesaid compared amylases at various pH levels. Rec. time: 30 minutes. Rec. temperature: 40' C.

FIG. 2 shows the activity of saccharifying power of the aforesaid compared amylases at various PH levels. Rec. time: 2 hours. Rec. temperature: 40 C.

FIG. 3 shows the stability of dextrinogenic power of the aforesaid compared amylases at pH 4.5, 40 C, for 30 minutes, after pre-treatment at various pH levels, 40 C, for 30 minutes.

FIG. 4 shows the stability of saccharifying power of the aforesaid compared amylases at pH 4.5, 40 C, for 30 minutes, afterpretreatment at various pH levels, 40 C, for 2 hours.

FIG. 5 shows the effect of rec. temperature on the dextrinogenic power of the aforesaid compared amylases. Rec. pH: 4.5. Rec. time: 30 minutes.

FIG. 6 shows the effect of rec. temperature on the saccharifying power of the aforesaid compared amylases. Rec. pH: 4.5 Rec. time: 2 hours.

FIG. 7 sets forth graphically the effect of temperature on the stability of dextrinogenic power of the aforesaid compared amylases, after pre-treatment at pH 6.0, at various temperatures, for 30 minutes.

FIG. 0 sets forth graphically the effect of temperature on the stability of saccharifying power of the aforesaid compared amylases, after pretreatment at pH 6.0, at various temperatures, for 2 hours.

FIGS. 9 to 12 inclusive sets forth comparative results obtained with amylase of this invention (Example 2, infra) vis-avis an amylase preparation derived from Paecilomyces varioti TPR-220.

FIG. 9 shows the comparative results re activity of dextrinogenic power at various pH levels. Rec. temperature: 40 C. Rec. time: 30 minutes.

FIG. 10 shows the comparative results re activity of saccharifying power at various pH levels. Rec. temperature: 40 C. Rec. time: 2 hours.

FIG. 11 shows the effect of temperature on the dextrinogenic power. Rec. pH: 4.5. Rec. time: 30 minutes.

FIG. 12 shows the effect of temperature on the saccharifying power. Rec. pH: 4.5. Rec. time: 2 hours.

EXPLANATION OF LEGENDS In FIGS. 1 to 8 inclusivem 0 represents the new amylase according to Example I O- 0 represents amylase preparation A represents amylase preparation B A- "A represents amylase preparation C [:l l:l represents amylase preparation D In FIGS. 9 to 12 inclusiverepresents the new amylase according to Example 2 represents amylase from Paecilomyces varioti TPR-220 INTERPRETIVE EXPLANATIONS 1. pH activity curves of amylase The digestive activity of the compared amylase preparations has been determined as shown in FIGS. 1 and 2. The amylase preparations derived from Paecilomyces subglobosum TPR-38 l0 and TPR-38ll possess dextrinogenic and saccharifying powers at pH ranging from about pH 2.5 to 6.0, in particular more excellent activity at pH ranging from pH 3.0 to 6.0, substantially stronger activities than the other compared preparations at pH ranging from 2.5 to 3.5 and their optimum pH approximately at pH 4.0 to 4.5.

On the other hand, the commercial amylase preparations A, B, C, D require pH 5.0 for optimum condition.

As result of these determinations, it is clear that the activity of the new amylase preparations of the present invention is more excellent than that of the compared commercial preparations under acidic conditions.

2. pH stability curves of amylase The dextrinogenic and saccharifying powers of the new amylase preparations are of good stability at pH ranging from about pH 3.5 to 9.0 in comparison with the above commercial amylase preparations which require pH 5 to 9.0 as shown in FIGS. 3 and 4.

Thus, the new amylase preparations show more excellent stability and activity under acidic conditions.

3. Optimum temperature of amylolytic activity at pH 4.0 to 4.5

New amylase preparations 50 C Commercial amylase preparations about 40 C 4. Heat stability of amylolytic activity at pH 4.0 to 4.5

All the commercial amylase preparations are stable at below 40 C but the new amylase preparations are more stable than the commercial amylase preparations at 50 C.

Comparative tests have also been made as to amylolytic activities and characteristics between the new strains and l aecilomyces varioti TPR-220, which have considerable morphological similarities.

The differences and superiority of the new amylase preparations are made clear by the following tables and facts, showing that the amylase preparations of this invention exhibit more excellent effects in dextrinogenic and saccharifying activities as shown in FIGS. 9 to 12.

5. Differences of the pH activity and active temperature PH activity Temperature activity Active Optimum New amylase range pH (Optimum temp.) (according to Ex.2) pH 2.5-7.0 pH 4.04.5 50'' C. New amylase pH2.5-'7.0 pH 4.0-4.5 50 C. (according to Ex.l) Amylase of Paecilo pH 3.0-7.0 pH 5.0 50-60 C. myce: vanori TPR-220 Dextrinogenic power Saccharifying power (at pH 4.0-4.5) (at pH 4.04.5) New amylase 70,000 unit/g. 1 10,000 unit/g. (according to Ex.2) New amylase 200,000 unit/g. 220,000 unit/g. (according to Ex.l Amylase of Paecilo 4,000 unit/g. 13,000 unit/g. mycea varioti TPR-ZZO AMYLOLYTIC ACTIVITIES Dextrinogenic power Saccharifying power pH 2.5 pH 3.0 pH 2.5 pH 3.0

unit/g. unit/g. unit/g. unit/g. New amylase 80,000 160,000 60,000 120,000 (according to Ex.l) Amylase preparation A 10,000 12,000 Amylase preparation B 5,000 10,000 Amylase preparation C 20,000 10,000 Amylase preparation D 4,000 7,000

CALCULATION OF DEXTRINOGENIC AND SACCHARIFYING POWERS The dextrinogenic and saccharifying powers were calculated by the following determinations.

i. Starch dextrinogenic power: assaying by blue value method I ml. of an amylase solution was added to 10 ml. of 2 percent soluble starch solution containing a half quantity of 1/10 M sodium citrate buffer solution and kept at 40C for 30 minutes.

Sampling up 1 ml. of the mixture, 1 ml. of l N-l-lCl was added to the mixture to stop the reaction, and further 10 ml. of N/ ,000 iodine solution were added.

The sample was assayed by optical density (wave length: 660 mp., absorption layer: 1 cm.).

A blank test was carried out by the same procedure using distilled water instead of the enzyme solution.

DEXTRINOGENIC POWER UNIT Blank value Assay value/Blank value) X 1,000 X 1/30 X 1,000 /n wherein n mg. of enzyme preparation used.

In this determination, the unit is based on dextrinogenic power necessary for decreasing the blue color reaction in ml. of 2 percent soluble starch to 0.1 percent per minute at 40 C., whereby the value is calculated as one unit and it is practically exchanged by the unit per g. of the used amylase preparation.

ii. Saccharifying power: assaying by iodine method 1. ml. of an amylase solution was added to 20 ml. of 2 percent soluble starch solution containing a half quantity of l/ 10 M sodium citrate buffer solution and kept at 40 C for 2 hours.

20 ml. of N/ 10 iodine solution and 20 ml. of N/S NaOl-l were added and then kept standing at room temperature.

After standing for 20 minutes, 4 ml. of l N-H SO were added and free iodine was titrated with 0.1 N-Na s O Similarily, a blank test was carried out by the same procedure where enzyme solution was not added, after addition of iodine solution.

This unit is based on the power necessary for saccharifying 20 ml. of 2 percent soluble starch at 40 C, whereby the value is calculated as one unit when 1 mg. of glucose is produced when using 1 g. of the amylase in iodine titration.

The following examples illustrate presently preferred exemplary embodiments of the invention, but are not intended as a limitation thereof.

EXAMPLE 1 lst step:

Liquid culture medium for seed koji Potato starch Peptone 60 3.

Potassium dihydrogen phosphate 4 g. Potassium chloride 2 g. Magnesium sulphate l g. Ferrous sul hate 0.2 g

Main liquid culture medium Potato starch 5 kg. (kilograml) Peptone 3 kg.

Potassium dihydrogen phosphate 200 g.

Potassium chloride l00 g. Magnesium sulphate 10 g.

Water l00 liters The above materials are mixed and adjusted to pH 6.0 with conc. HCl.

Similarly, the medium is sterilized at C for 10 minutes. The seed koji pre-incubated by 1st step is inoculated upon the main liquid culture medium and cultivated in the culturing tank under the following conditions: temperature: 30 C; aeration rate: liters/min; stirring: 350 r.p.m.; cultivating time: 94 hours.

Then the cultured medium is centrifuged to separate fungi and 60 liters of supernatant containing an acid-active and stable amylase are obtained with the centrifuging.

Thus-obtained supernatant is adjusted to pH 6.0 and 130 liters of 95 percent ethanol cooled at 20 C are added into the supernatant in order to obtain a precipitation of the amylase.

The precipitate is thoroughly washed with ethanol and dried in vacuo at 30 C.

180 g. of powdered amylase preparation are obtained from the preparation, which has 200,000-205,000 unit/g. in dextrinogenic power at pH 4.0 to 4.5 and 210,000-220,000 unit/g. in saccharifying power at pH 4.0 to 4.5.

EXAMPLE 2 1st step:

Solid culture medium for seed koji Wheat bran 500 g. Chall' 300 g. Water 400 ml.(milliliters) 2nd step:

Main solid culture medium Wheat bran 50 kg. Chat? 30 kg. Water 40 liters The above materialas are mixed to make the main solid culture medium base.

Similarly, the medium is sterilized at 125 C for 30 minutes.

The seed koji pro-incubated by 1st step is inoculated upon the main solid culture medium and layed to cultivate at 30 C for 70 hours.

250 liters of water are added to the cultured medium to extract the amylase.

After extracting at 30 C for 3 hours and further centrifuging, liters of transparent solution containing an acid-active and stable amylase are given.

Thus-obtained solution is mixed with 330 liters cent ethanol cooled at -20 tion of the amylase.

The precipitate is washed with ethanol and dried in vacuo at 30 C. 900 g. of powdered amylase preparation are obtained from the precipitate, which has 70,000 unit/g. in dextrinogenic power at pH4.0 to 4.5 and 110,000 unit/g. in saccharifying power at pH 4.0 to 4.5.

The acid-active and acid-stable amylases of the present invention are useful, by virtue of their superior amylolytic, dextrinogenic and saccharifying activities in extended fields, for example as digestive for medicaments, for pre-treatment of materials for processing foods, as enzymes for ripening materials during production, as clarificant of fermentative foods of 95 per- C in order to obtain a precipitasuch as beer, sake (Japanese wine), vinegar and the like, as a tenderizer of beef or corn before cooking, as well as for other purposes for which amylases are conventionally used. The instant amylases are however more especially suitable for use under acidic conditions and particularly as digestive enzyme which functions against dietary starch in the stomach where the gastric juice provides a strongly acidic condition.

A culture of Paecilamyces subglobosum, strain TPR-38ll is on deposit at American Type Culture Collection, Rockville, Maryland, under ATCC accession number 16492. A culture of Paecilomyces subglabosum, strain TPR-38 10 is similarly on deposit under ATCC accession number 16493.

What is claimed is: p

l. A process for the preparation of acid-active and stable amylase having superior amylolytic activities under acidic conditions, which comprises, in combination, the steps of lst: pre-incubating a microorganisms selected from the group of Paecilomyces subglobosu'm strain TPR-38 l (ATCC 16493) and Paecilamyces subglobosum strain TPR-38ll (ATCC 16492) on a sterilized liquid or solid culture medium for seed koji at 30C for 70 hours under aerobic conditions and thereby obtaining a seed koji of the Paecilomyces subglogosum group; and

2nd: inoculating and cultivating the thus-obtained seed koji on a main liquid or solid culture medium at 30C under aerobic conditions until the objective acid-active and stable amylase is substantially accumulated in the culture medium, and recovering the acid-active and stable amylase from the cultured medium.

2. A process according to claim 1, wherein the 2nd step cultivation is carried out for about 70 to 90 hours.

3. A process according to claim 1, wherein the microorganism is Paecilomyces subglobosum TPR-SB l0 (ATCC 16493).

45A process according to claim 1, wherein the microorganism is Paecilomyces subglobosum TPR38ll (ATCC 16492).

5. A process according to claim 1, wherein the main liquid culture medium and the liquid culture medium for seed koji are aqueous media and contain:

a. a carbon source selected from the group consisting of starch and maltose, in a total amount of 5 percent by weight;

b. a nitrogen source constituted by peptone in a total amount of 5 percent by weight; and

c. 0.05 percent of magnesium sulfate, 0.01 percent of ferrour sulfate, 0.1 percent of potassium chloride and 0.2 percent of potassium dihydrogen phosphate, all by weight.

6. A process according to claim 5 wherein the carbon source also comprises a member selected from the group consisting of glucose, dextrine, glycerol and wheat flour, the total amount of carbon source not exceeding the said 5 percent by weight, and wherein the nitrogen source also comprises a member selected from the group consisting of raw soybean flour, defatted soybean, dried yeast and corn steep liquor, the total amount of nitrogen source not exceeding the said 5 percent by weight.

7. A process according to claim 1, wherein the main solid culture medium or the solid culture medium for the seed koji is constituted by wheat bran, chaff and water in the following ratio:

wheat bran:chaff:water= 5:3:about 4 by weight.

8. In a process for the fermcntative production of amylase by the culturing of an amylase-producing microorganism in a nutrient medium therefor, the improvement according to which the microorganism is a member selected from the group consisting of Paecilomyces subglobosum strain TPR-38 l0 (ATCC 16493) and Paecilomyces subglobosum strain TPR-38ll (ATCC 16492) and, whereby the obtained amylase is characterized by the following:

a. amylolytic dextrinogenic and saccharifying activities in a broad pH range from FROM 2.5 to 6.0, said activities being superior to those of amylases from Aspergillus niger,

Aspergillus aureus, As rgillus oryzae and Paecilognyces variotz in the acidic p range from 2.5 to 3.5 the optimum pH ranging from 4.0 to 4.5,

b. stability of dextrinogenic and saccharifying powers in a broad pH range from 3.5 to 9.0,

c. 70,000 to 200,000 unit/g. in dextrinogenic power and 110,000 to 220,000 unit/g. in saccharifying power in the acidic pH range from 4.0 to 4.5; and

d. heat stability at'about 50C and effective amylolytic, dextrinogenic and saccharifying activity at 40C at pH ranging from 4.0 to 4.5. 

2. A process according to claim 1, wherein the 2nd step cultivation is carried out for about 70 to 90 hours.
 3. A process according to claim 1, wherein the microorganism is Paecilomyces subglobosum TPR-3810 (ATCC 16493).
 4. A process according to claim 1, wherein the microorganism is Paecilomyces subglobosum TPR-3811 (ATCC 16492).
 5. A process according to claim 1, wherein the main liquid culture medium and the liquid culture medium for seed koji are aqueous media and contain: a. a carbon source selected from the group consisting of starch and maltose, in a total amount of 5 percent by weight; b. a nitrogen source constituted by peptone in a total amount of 5 percent by weight; and c. 0.05 percent of magnesium sulfate, 0.01 percent of ferrous sulfate, 0.1 percent of potassium chloride and 0.2 percent of potassium dihydrogen phosphate, all by weight.
 6. A process according to claim 5 wherein the carbon source also comprises a member selected from the group consisting of glucose, dextrine, glycerol and wheat flour, the total amount of carbon source not exceeding the said 5 percent by weight, and wherein the nitrogen source also comprises a member selected from the group consisting of raw soybean flour, defatted soybean, dried yeast and corn steep liquor, the total amount of nitrogen source not exceeding the said 5 percent by weight.
 7. A process according to claim 1, wherein the main solid culture medium or the solid culture medium for the seed koji is constituted by wheat bran, chaff and water in the following ratio: wheat bran:chaff:water 5:3:about 4 by weight.
 8. In a process for the fermentative production of amylase by the culturing of an amylase-producing microorganism in a nutrient medium therefor, the improvement according to which the microorganism is a member selected from the group consisting of Paecilomyces subglobosum strain TPR-3810 (ATCC 16493) and Paecilomyces subglobosum strain TPR-3811 (ATCC 16492) and, whereby the obtained amylase is characterized by the following: a. amylolytic dextrinogenic and saccharifying activities in a broad pH range from 2.5 to 6.0, said activities being superior to those of amylases from Aspergillus niger, Aspergillus aureus, Aspergillus oryzae and Paecilomyces varioti in the acidic pH range from 2.5 to 3.5, the optimum pH ranging from 4.0 to 4.5; b. stability of dextrinogenic and saccharifying powers in a broad pH range from 3.5 to 9.0; c. 70,000 to 200,000 unit/g. in dextrinogenic power and 110,000 to 220,000 unit/g. in saccharifying power in the acidic pH range from 4.0 to 4.5; and d. heat stability at about 50*C and effective amylolytic, dextrinogenic and saccharifyIng activity at 40* C at pH ranging from 4.0 to 4.5. 